Multiprotein complexes that mediate crucial cellular functions, such as signal transduction and membrane traffic, are assembled at the interface of the membrane and the cytosol. The apposition of multiprotein complexes on membrane-cytosol interfaces is achieved in several ways. The best understood manner involves the anchoring of the complex around one or several integral membrane proteins, as is for example the case of complexes of receptor tyrosine kinases and cytosolic signaling proteins. Other multiprotein assemblies are anchored on the polar head groups of phosphoinositides. For example, coat proteins involved in membrane budding assemble onto phospholipid bilayers, and signaling complexes can specifically assemble onto 3' phosphoinositides. The molecular basis for the formation of such phospholipid-based assemblies is poorly understood. This Program Project combines biochemical, structural and imaging approaches to address the question of how multiprotein assemblies are organized around 3' phosphoinositides. The Program Project format is required to effectively combine divergent techniques, which include X-ray crystallography, live cell imaging using digital imaging microscopy with laser-based illumination, and powerful deconvolution algorithms to achieve resolution at the nanometer level. Two model systems will be studied. First, the Czech group will study subcellular localizations of PtdIns(3)P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 and the multiprotein signaling complexes composed of GRPI, ARF-GTPase, and GRSPI, which are anchored around Ptdins(3,4,5)P3. Second, the Corvera group will study an endosome fusion complex containing EEA1, RabS and calmodulin, which is anchored around PtdIns(3)P. Specific questions to be addressed include: is the interaction of a protein with a phospholipid head group necessary and sufficient to determine its localization to specific regions of the membrane? For this, GFP-fusions of the proteins cited above will be used to determine the subcellular distributions of 3' phosphoinositides in fixed and live cells relative both to the kinases that produce them and to the other components of the complex. As a further means to determine the mechanisms by which these complexes function, the Lambright group will seek to solve the crystal structures of individual protein components as well as of their complexes with lipid head groups. Such structures will be important in defining the spatial topographies of signaling complexes relative to the membranes to which they are bound. The activities in the three projects are highly integrated and collaborative, and are supported by an Imaging Core rated as outstanding in the previous review.